LC Tissue Distribution Study of Paeonol in Rats after Oral Administration

Paeonol, an important constituent of the traditional Chinese medicine Cortex Moutan, has a variety of bioactivity. A simple and sensitive HPLC?CUV method has been developed for analysis of paeonol in different rat tissue (heart, liver, spleen, lung, kidney, and brain). Bio-samples were prepared by simple protein precipitation, and separation of paeonol was achieved on a C₁₈ column with methanol?C2% glacial acetic acid solution 70:30 (v/v) as mobile phase at a flow rate of 1.0 mL min^?1. UV detection was at 274 nm and the column temperature was 30 °C. Linearity was good between 0.025 and 5.0 ??g mL^?1 (r ² ?? 0.9990) for tissue samples. Inter-day and intra-day accuracy (as relative error, RE) and precision (as relative standard deviation, RSD) were <5.94 and 6.05%, respectively. The limit of detection was 0.025 ??g mL^?1 and extraction recovery for all samples was ??85.86%. The method was successfully applied to a tissue-distribution study after oral administration of 40 mg kg^?1 paeonol to healthy Sprague?CDawley rats. The study showed that paeonol was quickly distributed and eliminated after oral administration; liver and kidney were the major distribution tissues of paeonol in rats, and paeonol quickly passed through the blood?Cbrain barrier. It was also found there was no long-term accumulation of paeonol in rat tissues.     

Rapid and Selective LC�CMS�CMS Method for the Determination of cyclo-trans-4-l-Hydroxyprolyl-l-serine (JBP485) in Rat Plasma

A rapid and selective liquid chromatographic/tandem mass spectrometric method for the determination of JBP485 was developed and validated. Following protein precipitation, the analyte and internal standard (JBP923) were separated from human plasma using an isocratic mobile phase on an Elite Kromasil C18 column. An API 3200 tandem mass spectrometer equipped with a Turbo ionSpray ionization source was used as the detector and operated in the positive ion mode. Multiple reaction monitoring using the precursor to product ion combinations of m/z 201.2 ?? 86.2 and m/z 219.2 ?? 86.2 was performed to quantify JBP485 and JBP923, respectively. The method was linear in the concentration range of 0.10?C50.00 ??g mL^?1 using 100 ??L of plasma. The lower limit of quantification was 0.10 ??g mL^?1. The intra- and inter-day relative standard deviations over the entire concentration range were less than 6.65%. Accuracy determined at three concentrations (0.25, 4.00 and 25.00 ??g mL^?1 for JBP485) ranged from ?0.78 to 2.74% in terms of relative error. Each plasma sample was chromatographed within 2.0 min. The method was successfully applied to characterize the pharmacokinetic profiles of JBP485 in rats after an intravenous injection of 6.25 mg kg^?1 JBP485.     

Development and Validation of a Stability-Indicating LC Method for Simultaneous Analysis of Aceclofenac and Paracetamol in Conventional Tablets and in Microsphere Formulations

A stability-indicating reversed-phase LC method for analysis of aceclofenac and paracetamol in tablets and in microsphere formulations has been developed and validated. The mobile phase was 80:20 (v/v) methanol–phosphate buffer (10 mM at pH 2.5 ± 0.02). UV detection was at 276 nm. The method was linear over the concentration ranges 16–24 and 80–120 μg mL^?1 for aceclofenac and paracetamol, respectively, with recovery in the range 100.9–102.22%. The limits of detection and quantitation for ACF were 0.0369 and 0.1120 μg mL^?1, respectively; those for PCM were 0.0631 and 0.1911 μg mL^?1, respectively.     

LC Determination of Icariside II in Rat Plasma and Tissues: Application to a Tissue Distribution Study

A sensitive and reliable reversed-phase liquid chromatography (RP-LC) with ultraviolet (UV) detection has been developed and validated for the quantification of Icariside II in rat plasma and tissues using Fermononetin as the internal standard. Protein precipitation and liquid?Cliquid extraction were utilized for plasma and tissue sample preparation, respectively. The analysis was successfully carried out on an Agilent SB-C₁₈ column (5 ??m, 4.6 × 250 mm) with the implementation of the following conditions: a mobile phase of phosphoric acid solution (0.1%, v/w)?CAcetonitrile (55:45, v/v), a flow rate of 1 mL min^?1, a column temperature of 25 °C and a detection wavelength of 270 nm. Good linear relationships of calibration curves were obtained (r ² > 0.9906) over the investigated concentration range with plasma and tissue samples. The lower limit of quantification (LLOQ) and the limit of detection (LOD) were 0.1 and 0.02 ??g g^?1, respectively (for plasma sample, they were 0.05 and 0.1 ??g mL^?1, respectively). The developed method which was embodied with good precision, accuracy, recovery and stability was corroborated to satisfy the requirements for biomedical sample analysis. This method has been successfully applied to tissue distribution study of Icariside II in rats after a single intravenous dose at 12.5 mg kg^?1. Results suggested that Icariside II was distributed to rat tissues rapidly with greater initial concentrations in kidney, lung and liver. Moderate initial distributions were obtained in rat muscle, heart, bone, spleen and plasma. Low amount of Icariside II was detected in testes, and no Icariside II could be detected in the brain.     

Separation and Determination of Amitriptyline and Nortriptyline in Biological Samples Using Single-Drop Microextraction with GC

The combination of liquid phase microextraction (LPME) based on a single drop and gas chromatography flame ionization detector (GC-FID) was used for separation and determination of amitriptyline and nortriptyline in human plasma and urine samples. The sample solution was kept alkaline (pH 12), then a microdrop of organic solvent (isooctane) was suspended in the donor solution; after extraction, the organic microdrop was injected into the GC-FID. Experimental LPME conditions were optimized. Finally, the enrichment factors (89.5?C139.0), the relative standard deviation (RSD%, n = 5) 1.1?C8.5, linearity ranges (0.05?C20 ??g mL^?1), and the limits of detections (0.01, 0.02 ??g mL^?1) for selected drugs were evaluated.     

Rapid and Simultaneous Determination of Efavirenz, 8-Hydroxyefavirenz, and 8,14-Dihydroxyefavirenz Using LC�CMS�CMS in Human Plasma and Application to Pharmacokinetics in Healthy Volunteers

We developed a rapid and sensitive method for determining efavirenz, 8-hydroxyefavirenz, and 8,14-dihydroxyefavirenz in human plasma simultaneously using liquid chromatography?Ctandem mass spectrometry (LC?CMS?CMS). Three compounds and ritonavir, an internal standard, were extracted from plasma using ethyl acetate in the presence of 0.1 M sodium carbonate after incubation of ??-glucuronidase (500 U). After drying the organic layer, the residue was reconstituted in mobile phase (acetonitrile:20 mM ammonium acetate, 90:10, v/v) and injected onto a reversed-phase C₁₈ column. The isocratic mobile phase was eluted at 0.2 mL min^?1. The ion transitions monitored in multiple reaction-monitoring mode were m/z 314 ?? 244, 330 ?? 258, 346 ?? 262, and 721 ?? 296 for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The retention time is 1.93, 1.70, 1.52, and 1.82 min for efavirenz, 8-hydroxyefavirenz, 8,14-dihydroxyefavirenz, and ritonavir, respectively. The coefficients of variation of the assay precision were less than 10.7%, and the accuracy was 90?C111%. The lower limits of quantification (LLOQ) were 5 ng mL^?1 for efavirenz and 8-hydroxyefavirenz. This method was used to measure the plasma concentrations of efavirenz and its metabolites from healthy volunteers after a single 600 mg oral dose of efavirenz. This analytical method is a very rapid, sensitive, and accurate to determine the pharmacokinetic profiles of efavirenz including its metabolites.     

Determination of Mildronate in Human Plasma and Urine by UPLC�CPositive Ion Electrospray Tandem Mass Spectrometry

A simple, rapid, and selective method to determine the concentration of mildronate in human plasma and urine using ultra performance liquid chromatography?Ctandem mass spectrometry (UPLC-MS-MS) was developed and validated. The detection was performed on a triple-quadrupole tandem mass spectrometer by multiple reaction monitoring mode via electrospray ionization at m/z 147.2?C58.0 for mildronate and m/z 147.2?C87.8 for the internal standard, carbachol. The UPLC separation was carried out with a UPLC BEH HILIC column. The mobile phase consisted of 0.08% formic acid in 30 mM ammonium acetate solution and acetonitrile (23:77, v/v). Plasma samples were extracted from plasma by protein precipitation and urine samples were diluted with the mobile phase. The analysis time was 3.5 min for each sample. Linear calibration curves ranged from 0.10 to 100.00 ??g mL^?1 in human plasma and 0.50 to 600.00 ??g mL^?1 in urine. The method had been successfully applied to a pharmacokinetic study in healthy volunteers. After single intravenously administration of 250, 500, and 750 mg mildronate, the elimination half-life (t _1/2) were (2.74 ± 0.67), (4.86 ± 0.82) and (5.16 ± 0.77) h, respectively. The t _1/2 for the 250 mg dose did vary significantly with other dosages (P < 0.05), mildronate may have non-linear pharmacokinetics in humans.     

Extraction of Azole Antifungal Drugs from Milk and Biological Fluids Using a New Hollow Fiber Liquid-Phase Microextraction and Analysis by GC-FID

A simple, rapid and sensitive high-performance liquid chromatography LC (HPLC) method of determining the concentration of the novel betulinic acid derivative DRF-4012 (5??-chloro-2,3-didehydroindolo[2??,3??:2,3]betulinic acid) in rat plasma for pharmacokinetic and toxicokinetic purposes has been developed and validated. A simple and fast protein precipitation was performed, and then an extraction using an ethyl acetate:methanol (75:25 v/v) mixture was used to extract DRF-4012 and an internal standard (IS, DRF-4015) from rat plasma. Chromatographic separation was achieved using a Zorbax Eclipse XDB-C8 reversed-phase column with UV detection at 235 nm. The isocratic mobile phase, phosphate buffer (water adjusted to pH 3 with 20% o-phosphoric acid) and acetonitrile (15:85, v/v), was run at a flow rate of 1.2 mL min^?1. The assay was linear (r ² > 0.99) over the concentration range 0.040?C75.0 ??g mL^?1, and presented limits of detection and quantification of 0.020 and 0.040 ??g mL^?1, respectively, in rat plasma. The absolute recovery of both the analyte and the IS was >85% from rat plasma. The intraday accuracy ranged from 99.25 to 102.67% with a precision of 2.62?C4.48%, and the interday accuracy ranged from 98.48 to 104.56% with a precision of 3.87?C5.68%. This developed and validated method was successfully used to determine the DRF-4012 concentration in rat plasma for a pharmacokinetic and toxicokinetic study after the intravenous administration of a 1 mg mL^?1 DRF-4012 nanoparticle formulation at doses of 2?C10 mg kg^?1 in Wistar rats.     

Determination of Dutasteride by LC: Validation and Application of the Method

A simple, rapid, and stability-indicating reversed-phase high-performance liquid chromatographic (LC) method for analysis for dutasteride has been successfully developed. Chromatography was performed on a 150 mm × 4.6 mm C₁₈ column with acetonitrile–water 60:40 (v/v) as isocratic mobile phase at 1.0 mL min^?1. Ultraviolet detection of dutasteride was at 210 nm. Its retention time was approximately 10 min and its peak was symmetrical. Response was a linear function of concentration over the range 0.2–1 μg mL^?1 (R ² = 0.997) and the limits of detection and quantitation were was 0.05 and 0.10 μg mL^?1, respectively. The method was validated for linearity, precision, repeatability, sensitivity, and selectivity. Selectivity was validated by subjecting dutasteride stock solution to photolytic, acidic, basic, oxidative, and thermal degradation. The peaks from the degradation products did not interfere with that from dutasteride. The method was used to quantify dutasteride in pharmaceutical preparations.     

Simultaneous Determination of Ibuprofen and Diphenhydramine Citrate in Tablets by Validated LC

A stability-indicating LC method was developed for the simultaneous determination of ibuprofen and diphenhydramine citrate in pharmaceutical dosage forms. The chromatographic separation was achieved on an Inertsil ODS 3V, 150 × 4.6 mm, 5 μm, column. The mobile phase contained a mixture of 50 mM potassium dihydrogen phosphate buffer:acetonitrile:triethylamine:glacial acetic acid (55:45:0.2:0.2, v/v/v/v). This method allowed the determination of 2.85–9.14 mg mL^?1 of ibuprofen and 0.54–1.73 mg mL^?1 of diphenhydramine citrate, in a diluent consisting of pH 7.2, 50 mM potassium dihydrogen phosphate buffer:acetonitrile (40:60, v/v). The flow rate was 1.2 mL min^?1 and the detection wavelength was 260 nm. The limit of detection for ibuprofen and diphenhydramine citrate was 1.72 and 0.54 μg mL^?1 and the limit of quantification was 5.73 and 1.64 μg mL^?1, respectively. This method was validated for accuracy, precision and linearity. The method was also found to be stability indicating.     

Simultaneous Quantification of Oroxylin A and Its Metabolite Oroxylin A-7-O-Glucuronide: Application to a Pharmacokinetic Study in Rat

A sensitive liquid chromatography?Celectrospray ionization?Ctandem mass spectrometry (LC?CESI?CMS?CMS) method was developed and validated for the quantification of cepharanthine (CEP) in beagle dog plasma. The chromatographic separation was performed on an Agilent-C18 column and the mobile phase was composed of methanol:water with 10 mM ammonium acetate (20:80, v/v). Detection was operated in the positive ion mode and the tandem mass spectrometer was tuned in the multiple reactions monitoring mode (MRM) to monitor m/z transitions 607 ?? 365 for CEP and 285 ?? 193 for the internal standard (IS) diazepam. This method exhibited a linear range of 5?C2,500 ng mL^?1. The precision (RSD%) and accuracy (RME%) of the assay were <8.7 and 2.4%, respectively. The limit of quantification was 5 ng mL^?1 and no significant matrix effect was observed. The validated method has been successfully applied to pharmacokinetic study of CEP in beagle dog.     

A Validated LC Method for the Quantitation of Cefotaxime in pH-Sensitive Nanoparticles

A sensitive and rapid routine LC method was validated for measuring cefotaxime incorporated in three different pH-sensitive nanoparticles. The drug was chromatographed on a C18 reversed-phase column; the mobile phase used was 0.05 M aqueous ammonium acetate, acetonitrile and tetrahydrofuran (87:11:2, v/v) adjusted to pH 5.5 with acetic acid. The flow rate was 1 mL min^?1 and cefotaxime was quantified at 254 nm, with a sensitivity range of 0.005 AUFS. The validated method was specific, linear (R ² ≥ 0.999), precise and accurate in a concentration range of 0.2–50.0 μg mL^?1. The method was rapid, selective and suitable for evaluation of cefotaxime in pH-sensitive Eudragit nanoparticles.     

LC Method for Quantification of ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic Acid in Rabbit Plasma: Validation and Application to a Pharmacokinetic Study

ent-11α-Hydroxy-15-oxo-kaur-16-en-19-oic acid (5F), a diterpenoid isolated from the Chinese herb Pteris semipinnata L, has been suggested to show antitumor properties. A simple and sensitive LC method was developed for the determination of 5F in rabbit plasma. The method involved liquid–liquid extraction using ethyl acetate under acidic conditions using naproxen as an internal standard. Separations were performed on a reversed-phase column with a mixture of 1% (v/v) glacial acetic acid and methanol (45:55, v/v) as mobile phase and UV detection was utilized at 242 nm. The calibration plot was linear in the range 0.20–10.0 μg mL^?1 (correlation coefficients r ²  > 0.998). The detection limit was 0.20 μg mL^?1, mean extraction recovery was above 82%, intra-day precision of the method was less than 6.4%, and inter-day precision was better than 8.7%, respectively. The validated assay was found to be suitable for the pharmacokinetic study of 5F in rabbits.     

Determination of Rizatriptan in Human Plasma by Liquid Chromatography Stable Isotope Dilution Electrospray MS�CMS for Application in Bioequivalence Study

A simple, sensitive, selective, rapid, rugged, reproducible and specific liquid chromatography?Ctandem mass spectrometry (LC?CMS/MS) method was used for quantitative estimation of rizatriptan (RZ) in human plasma using rizatriptan-d ₆ (RZD6) as internal standard (IS). Chromatographic separation was performed on Ascentis Express RP Amide C18, 50 × 4.6 mm, 2.7 ??m column with isocratic mobile phase composed of 10 mM ammonium formate:acetonitrile (20:80 v/v) at flow rate of 0.5 mL min^?1. RZ and RZD6 were detected with proton adducts at m/z (amu) 270.2 ?? 201.2 and 276.1 ?? 207.1, respectively, in multiple reaction monitoring (MRM) positive mode. Liquid?Cliquid extraction was used and validated over a linear concentration range of 0.1?C100.0 ng mL^?1 with correlation coefficient r ² ?? 0.9981. The limit of quantification (LOQ) and limit of detection (LOD) were found to be 0.1 ng mL^?1 and 12.5 fg, respectively. Intra- and inter-day precision were within 1.7?C3.1% and 2.8?C3.7%, and accuracy within 96.0?C101.7% and 99.7?C101.4% for RZ. Drug was found to be stable throughout three freeze?Cthaw cycles. The method was successfully employed for analysis of plasma samples following oral administration of RZ (10 mg) in 25 healthy Indian male human volunteers under fasting conditions.     

Simultaneous Determination of the Endogenous Free ��-Lipoic Acid and Dihydrolipoic Acid in Human Plasma and Erythrocytes by RP-HPLC with Electrochemical Detection

A highly sensitive, precise, and accurate reversed-phase high-performance liquid-chromatography/electrochemical detection method for simultaneous determination of the endogenous free ??-lipoic acid and dihydrolipoic acid in biological matrices was developed and validated. The two analytes were extracted from the samples with acetonitrile/10% metaphosphoric acid solution_(aqueous) (50/50 v/v). To determine the total lipoic acid, samples were treated with tris(2-carboxyethyl)phosphine solution in phosphate buffer, pH 2.5 with 85% orthophosphoric acid prior to deproteination. The two analytes were separated on a C₁₈ (150 × 4.6 mm, 5 ??m) analytical column using acetonitrile-50 mM phosphate buffer, pH 2.5 with 85% orthophosphoric acid (35/65 v/v) as the isocratic mobile phase pumped at a flow rate of 2.0 mL min^?1 at the column oven temperature of 35 °C. The column eluents were monitored at a potential of 0.9 V. These analytes were efficiently resolved in <7 min. The present method was sufficiently robust and specific for simultaneous determination of the two analytes and demonstrated acceptable values for linearity (r ² = 0.999 in the range of 0.1?C500 and 0.25?C1,000 ng mL^?1 for ??-lipoic acid and dihydrolipoic acid, respectively), recovery (>97%), precision (RSD% <2), and sensitivity (on column limit of detection, 150 and 375 fg for ??-lipoic acid and dihydrolipoic acid, respectively and limit of quantification: 0.5 and 1.25 pg for ??-lipoic acid and dihydrolipoic acid, respectively), indicating that the proposed method was more sensitive, precise, economical, and versatile, and has higher throughput than the previously reported methods for simultaneous determination of the two analytes.     

Determination of Tetrandrine in Rat Whole Blood by RP-LC and Its Application to Pharmacokinetic Studies

A simple, specific and sensitive RP-LC method was developed and validated for the determination of tetrandrine in rat whole blood for the first time. Chromatographic separation was performed on a Welchrom^TM C18 analytical column at a flow rate of 1.0 mL min^?1, using a mixture of methanol-water containing 0.6% triethylamine and 0.16% phosphoric acid as mobile phase. The wavelength for UV detection was set at 225 nm. Sample preparation involved a liquid-liquid extraction using n-hexane. The calibration curve was linear with r ² > 0.99 over a concentration range of 0.03?C6.4 ??g mL^?1 in rat whole blood with a lower limit of quantification of 0.03 ??g mL^?1. The intra- and inter-day precisions were 1.33?C4.55 and 3.33?C4.65%, respectively, and the intra- and inter-day accuracy ranged from ?5.24 to 0.90% and ?1.05 to 0.63%, respectively. No endogenous compounds were found to interfere with the analytes. Tetrandrine was stable for 8 h at room temperature, 24 h at 4 °C in rat whole blood, and for 30 days at ?20 °C after being prepared. For the first time, the present method was successfully applied to the pharmacokinetic studies of tetrandrine in rats after intravenous administration of three different doses. The results indicated that the pharmacokinetics of tetrandrine on rats was a first-order process.     

Determination of Quinine and Doxycycline in Rat Plasma by LC�CMS�CMS: Application to a Pharmacokinetic Study

A fast, sensitive, and specific LC?CMS?CMS method for determination of quinine (QN) and doxycycline (DOX) in rat plasma has been developed and validated. QN, DOX, and cimetidine (internal standard, IS) were extracted from the plasma by protein precipitation. The compounds were separated on a C₁₈ column with methanol?C0.1% aqueous formic acid 70:30 (v/v) as mobile phase at a flow rate of 0.5 mL min^?1 (split 1:3). Detection was by positive electrospray ionization (ESI+) in multiple reaction monitoring (MRM) mode, monitoring the transitions 325.0 ?? 307.0, 445.0 ?? 428.1, and 252.8 ?? 159.0, for QN, DOX, and IS, respectively. The analysis was carried out in 2.0 min and the method was linear in the plasma concentration range 5?C5,000 ng mL^?1. The mean extraction recoveries for QN, DOX, and IS from plasma were 89.4, 90.5, and 86.3%, respectively. The method was validated for linearity, precision, accuracy, specificity, and stability; the results obtained were within the acceptable range. The proposed method was successfully applied to the determination of QN and DOX in rat plasma samples to support pharmacokinetic studies.     

HILIC-MS-MS for the Quantification of Pidotimod in Human Plasma

A method for fast, sensitive, and specific hydrophilic interaction chromatography combined with tandem mass spectrometry (HILIC-MS/MS) was developed for the first time to determine the level of pidotimod in human plasma. With rosiglitazone as internal standard, analysis was carried out on a HILIC column (150 mm × 2.1 mm, 3.5 ??m) using a mobile phase consisting of methanol:0.2% formic acid (60:40, v/v). Detection was carried out by tandem mass spectrometry using electrospray ionization (ESI). Linear calibration curves were obtained in the concentration range of 11.2?C1.12 × 10⁴ ng mL^?1 for pidotimod, with a lower limit of quantification of 11.2 ng mL^?1. The intra- and inter-day precision values were high, with standard deviations lower than 15%, and the accuracy, in terms of relative error, ranged from ?10.5 to 9.4% at all quality control (QC) levels.     

Development and Validation of a Stability-Indicating LC Method for the Determination of Rupatadine in Pharmaceutical Formulations

A reversed-phase liquid chromatography (RP-LC) method was validated for the determination of rupatadine in pharmaceutical dosage forms. The LC method was carried out on a Gemini C₁₈ column (150 mm × 4.6 mm I.D.), maintained at 30 °C. The mobile phase consisted of ammonium acetate buffer (pH 3.0; 0.01 M) with 0.05% of 1-heptanesulfonic acid–acetonitrile (71.5:28.5, v/v), run at a flow rate of 1.0 mL min^?1 and using photodiode array (PDA) detection at 242 nm. The chromatographic separation was obtained with retention time of 5.15 min, and was linear in the range of 0.5–400 μg mL^?1 (r ² = 0.9999). The specificity and stability-indicating capability of the method was proven through the degradation studies and showing also, that there was no interference of the excipients. The accuracy was 100.39% with bias lower than 0.58%. The limits of detection and quantitation were 0.01 and 0.5 μg mL^?1, respectively. Moreover, method validation demonstrated acceptable results for precision, sensitivity and robustness. The proposed method was applied for the analysis of pharmaceutical dosage forms assuring the therapeutic efficacy.     

Simultaneous Determination of Zoalene and Its Metabolite in Chicken Muscle and Liver by SPE and UPLC�CMS-MS

This paper presents an analytical method for the simultaneous determination of zoalene and its metabolite 3-amino-5-nitro-o-toluamide (3-ANOT) in chicken muscle and liver by solid phase extraction and UPLC?CMS-MS operated in the positive and negative ionization switching mode. Samples were extracted with phosphate buffer solution and purified with OASIS^? HLB cartridge after pH adjustment. The determination was carried out using UPLC?CMS-MS on a Waters Acquity BEH C₁₈ column with 0.1% formic acid in water/acetonitrile as mobile phase with gradient elution. The linearity of the analytical response across the studied range of concentrations (2.0?C1,000 ??g L^?1) was excellent, obtaining correlation coefficients higher than 0.999. Matrix effects had been investigated for zoalene and 3-ANOT. Recovery studies were carried out on spiked chicken muscle and liver blank samples, at four concentration levels (50, 1,500, 3,000, and 4,500 ??g kg^?1 for chicken muscle and 50, 3,000, 6,000, and 9,000 ??g kg^?1 for chicken liver) performing six replicates at each level. Mean recoveries of 77.9?C94.2% with CVs of 3.2?C8.7% were obtained. The method demonstrated to be suitable for the simultaneous determination of zoalene and 3-ANOT in chicken tissues.